Human milk permeate compositions and methods of making and using same

ABSTRACT

This disclosure features human milk permeates and compositions containing the same obtained from fractionated whole human milk. The oligosaccharide rich permeate and permeate compositions of the present invention are useful as nutritional supplements for pre-term and full term infants, for establishing or maintaining gut flora and for treating the symptoms of inflammatory bowel disease.

This application claims priority to U.S. Provisional Application No.61/193,546, filed Dec. 5, 2008 and U.S. Provisional Application No.61/119,176, filed Dec. 2, 2008, both of which are incorporated herein intheir entirety.

TECHNICAL FIELD

This disclosure is related to human milk permeate compositions, e.g.,compositions comprising human milk oligosaccharides, and methods ofmaking and using such compositions.

BACKGROUND

Human milk is generally the food of choice for preterm and full terminfants because of its nutritional composition and immunologic benefits.The source of human milk can be, e.g., a donor or the infant's mother.The nutritional value of raw or conventionally-processed donor milk,however, varies and, in most instances, is not sufficient to meet theneeds of preterm infants. In addition, a possibility of bacterial, viraland other contamination of raw donor milk exists. Even the mother's ownmilk is often not nutritionally sufficient for the premature infant. Itis often desirable to feed preterm infants milk that is fortified withvarious supplements, e.g., oligosaccharides. Compositions that containhuman milk oligosaccharides can also be useful as supplements forinfants, children, and adults who have or are at risk of developingvarious disorders and diseases, e.g., infections or immune deficiencies,and who need to develop and/or maintain proper gut flora.

SUMMARY

This disclosure features human milk permeate compositions, e.g.,compositions that include human oligosaccharides, peptides, and othersmall molecules, and methods of making and using such compositions. Thepermeate compositions can contain various levels of nutritionalcomponents and can be used in feeding preterm and full term infants, aswell as children and adults with various disorders and/or diseases. Thecompositions are generated from, inter alia, filtered portions of humanmilk. The present inventors have found that surprisingly, permeate(which had been thought to be a waste product lacking significantnutritional value) contains high biologically active content, includinghuman oligosaccharides. It was found that the oligosaccharide content ofthe permeate and the human milk products described in application U.S.Ser. No. 11/947,580 (2008/0124430), both natural and concentrated, didnot differ substantially with respect to size and composition ascompared to mother's milk. Because the starting material, from whichpermeate is obtained, is pooled human milk, permeate and other processedhuman milk products (e.g., those described in application U.S. Ser. No.11/947,580) can contain more varieties of oligosaccharides thanindividual mother's milk. Therefore, the permeate can be added tonon-human (e.g. bovine) and/or human milk to increase its nutritionaland/or immunologic value. The permeate can also be used to fightinfections, treat inflammatory bowel disease and help develop andmaintain proper gut flora. The permeate can also be diluted orconcentrated and used in such forms as a nutritional supplement. Similarbenefits can be obtained from permeate obtained by processing pooledhuman milk and administering it to infants, e.g., premature infants(e.g., pooled human milk described in application U.S. Ser. No.11/947,580).

The methods featured herein are used to process large volumes of donormilk, e.g., about 75-2,000 liters/lot of starting material.

In a first aspect, the present invention provides a compositioncomprising milk and a human milk permeate. In one embodiment, the milkis human milk. In another embodiment, the milk is non-human milk. Insome embodiments, the non-human milk is bovine milk. In someembodiments, the permeate of the composition is obtained by filteringliquid from human milk. In some embodiments, the composition is obtainedby filtering liquid from human skim milk. In some embodiments, thecomposition further comprises vitamins and minerals.

In a second aspect, the present invention provides a nutritionalcomposition comprising a concentrated or diluted human milk permeate. Inone embodiment, the permeate is obtained by filtering liquid from humanmilk. In other embodiments, the permeate is obtained by filtering liquidfrom human skim milk. In some embodiments, the composition furthercomprises vitamins and minerals.

In a third aspect, the present invention provides a method of making aconcentrated or a diluted human milk permeate, comprising (a) obtaininghuman milk, (b) separating the milk into cream and skim; (c) filteringthe skim to obtain a permeate; (d) retaining the permeate; and (e)diluting, concentrating or drying the permeate. In some embodiments thediluting step in (e) comprises adding a non-human milk to the permeate.In some embodiments, the non-human milk is bovine milk. In other aspectsof the invention, the diluting step in (c) comprises adding water or abuffer to the permeate. In some embodiments, the concentrating step in(e) comprises reverse osmosis. In some embodiments, the method of makinga concentrated or diluted human milk permeate further comprises addingvitamins and minerals to the permeate after step (d). In someembodiments, the filtering in step (c) comprises ultrafiltration. Insome embodiments, the method of making a concentrated or diluted humanmilk permeate further includes (f) reducing the bioburden. In someembodiments, reducing the bioburden comprises pasteurization or sterilefiltration.

In a fourth aspect, the present invention provides a method ofadministering a permeate composition to a subject comprising human milkoligosaccharides to a subject comprising (a) obtaining human milk; (b)separating the milk into cream and skim; (c) filtering the skim toobtain a permeate; (d) retaining the permeate; and (e) administering thepermeate to the subject. In some embodiments, the subject is a humanpreterm or full term infant. In some embodiments, the composition isadministered topically or orally. In some embodiments, the compositionis administered orally via a feeding tube.

In a fifth aspect, the present invention provides a method ofestablishing beneficial gut flora in a subject comprising administeringa permeate composition comprising oligosaccharides from human milk to asubject. In some embodiments, the subject is a human. In someembodiments, the subject is a human preterm or full term infant. In someembodiments, the human preterm or full term infant is fed formula afterbirth. In some embodiments, the composition is administered prior to,concurrently with, or following other compositions useful forestablishing beneficial gut flora. In some embodiments, the othercompositions useful for establishing beneficial gut flora are probioticbacteria or plant polysaccharides. In some embodiments, the compositionis administered in conjunction with a non-human milk formulation. Insome embodiments, the composition is in a mixture with a non-human milkformulation. In some embodiments, the composition is a human milkultrafiltration permeate. In some embodiments, the permeate is obtainedby filtering human milk. In some embodiments, the permeate is obtainedby filtering human skim milk. In some embodiments, establishingbeneficial gut flora comprises populating the gut with bifidobacteria orlactobacilli or both.

In a sixth aspect, the present invention provides a method of treating asubject who has an infection or is at risk of developing an infectioncomprising administering a permeate composition comprisingoligosaccharides from human milk to the subject. In some embodiments,the symptoms of the infection are caused by bacteria, bacterial toxinsor viruses. In some embodiments, the subject is a human. In someembodiments, the subject is a human neonate, infant, child or an adult.In some embodiments, treating comprises ameliorating at least onesymptom of the infection. In some embodiments, treating comprisespromoting the development of beneficial gut bacteria. In someembodiments, the beneficial gut bacteria are bifidobacteria orlactobacilli or both.

In a seven aspect, the invention provides a method of treating a subjectsuffering from an inflammatory bowel disease, the method comprisingadministering a permeate composition comprising oligosaccharides fromhuman milk to a subject. In some embodiments, the inflammatory boweldisease is one or more of Crohn's disease, irritable bowel disease,ulcerative colitis (UC), indeterminate colitis, microscopic colitis,collagenous colitis and pseudomembrenous colitis. In some embodiments,the subject is a human. In some embodiments, the subject is a humanneonate, infant, child or an adult. In some embodiments, treatingcomprises ameliorating at least one symptom of the inflammatory boweldisease. In some embodiments, treating comprises promoting developmentof beneficial gut bacteria. In some embodiments, the beneficial gutbacteria are bifidobacteria or lactobacilli or both.

In an eighth aspect, the invention provides a method of making anutritional supplement comprising obtaining a permeate compositioncomprising oligosaccharides from human milk and supplementing anon-human milk formulation with the composition. In some embodiments,the composition is a concentrated or diluted human milk permeatecomposition. In some embodiments, the permeate composition is obtainedby filtering liquid from human milk. In some embodiments, the permeatecomposition is obtained from filtering liquid from the skim portion ofhuman milk.

In a ninth aspect, the invention provides a method of making aconcentrated or diluted human milk permeate comprising (a) obtaininghuman milk; (b) separating the milk into cream and skim; (c) filteringthe skim to obtain a permeate; (d) retaining the permeate; and (e)diluting, concentrating, or drying the permeate. In some embodiments,the diluting step (e) comprises adding a non-human milk composition tothe permeate. In some embodiments, the non-human milk is bovine milk. Insome embodiments, the diluting step in (e) comprises adding deionizedwater or a buffer. In some embodiments the concentrating step in (e)comprises reverse osmosis. In some embodiments, the method furthercomprises adding vitamins and minerals to the permeate after step (d).In some embodiments, the filtering in step (c) comprises ultrafiltering.In some embodiments, the method further comprises (f) reducing thebioburden. In some embodiments, reducing the bioburden comprisespasteurization or sterile filtration.

All patents, patent applications, and references cited herein areincorporated in their entireties by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart of an embodiment of a method of making human milkpermeate.

FIG. 2 is a chart showing a summary of oligosaccharide composition ofsamples 42, 48, 49, 53, and 54.

FIG. 3 is a histogram of common human milk oligosaccharides found inhuman milk and in samples 42, 48, 54 (all bulk final product), 53(ultrafiltration permeate), and 49 (initial pool of donor milk).

FIG. 4 is a graph showing a typical mass spectrometric profile of freeoligosaccharides in human milk. Round dots represent human milkoligosaccharides.

FIG. 5 is a graph showing mass spectrum profile obtained for sample 42(PROLACT+™ H2MF™) (See U.S. Ser. No. 11/947,580) Round dots representhuman milk oligosaccharides.

FIG. 6 is a graph showing mass spectrum profile obtained for sample 53(PROLACT+™ H2MF™ process byproduct or permeate) (See U.S. Ser. No.11/947,580). Round dots represent human milk oligosaccharides.

FIG. 7 is a graphical representation of the process of separatingpermeate from collected whole human milk.

DETAILED DESCRIPTION

This disclosure features human milk permeate compositions, e.g.,compositions that include human oligosaccharides, peptides, and othersmall molecules and methods of making and using such compositions. Thecompositions contain various levels of nutritional components and can beused in feeding of or administration to preterm and full term infants,as well as children and adults with various disorders and/or diseases.The compositions are generated from, inter alia, filtered portions ofhuman skim milk. The present inventors have found that, surprisingly,permeate (which had been thought to be a waste product lackingsignificant nutritional value) contains high biologically activecontent, including human oligosaccharides. Because the startingmaterial, from which permeate is obtained, is pooled human milk,permeate can contain more discrete molecular forms or types ofoligosaccharides than individual mother's milk.

The permeate can be added to non-human or human milk to increase itsnutritional value. For example, the permeate can be added to human milkfortifiers and standardized milk compositions described in applicationU.S. Ser. No. 11/947,580, filed on Nov. 29, 2007, the contents of whichare incorporated herein by reference in their entirety. The permeate canalso be added to non-human milk formula, e.g., bovine milk formulationsor mixtures of human and non-human milk formulations. Without beingbound by theory, it is believed by the inventors that permeatecompositions comprising human milk oligosaccharides, peptides, and othersmall molecules can be used to promote development of beneficial gutflora in preterm infants or neonates and to maintain proper gut flora inchildren and adults. Permeate can also be useful as a supplement forinfants, children, and adults who have or are at risk of developingvarious disorders and diseases, e.g., infections or immune deficiencies.The permeate can be diluted or concentrated and/or fortified withvitamins and minerals and used in such forms as a nutritionalsupplement.

The methods featured herein are used to process large volumes of donormilk, e.g., about 75-2,000 liters/lot of starting material. Thecompositions of the present disclosure are generated from human donormilk, e.g., pooled milk, which undergoes rigorous genetic screening andprocessing (e.g., to reduce the bioburden).

By “permeate” (also referred to herein as “permeate composition” or a“milk processing byproduct” or “milk byproducts”) is meant a portion ofmilk that has been processed by filtration, e.g., ultrafiltration, ofhuman milk, e.g. skim milk. Typically, the screen size used inultrafiltration is 1 Kda-1,000 Kda in size. The liquid passing throughthe filtration contains a significant amount of oligosaccharides and isreferred to as permeate.

By “whole milk” is meant milk from which no fat has been removed.

By “skim milk” is meant milk from which at least 75% of fat has beenremoved.

The terms “premature”, “preterm” and “low-birth-weight (LBW)” infantsare used interchangeably and refer to infants born less than 37 weeksgestational age and/or with birth weights less than 2500 gm.

The term “full term” infant is used to refer to infants born after 37weeks gestational age and/or with birth weights greater than 2500 gm.

By “bioburden” is meant microbiological contaminants and pathogens(generally living) that can be present in milk, e.g., viruses, bacteria,mold, fungus and the like.

Permeate Compositions and Methods of Obtaining Said Compositions

Permeate compositions featured herein are obtained from human milk,e.g., pooled donor milk. Methods of obtaining and screening human donormilk (including qualifying donors) are described in applications U.S.Ser. No. 11/947,580, and U.S. Ser. No. 11/526,127 (2007/0098863), thecontents of which are incorporated herein by reference in theirentirety.

FIG. 1 shows one embodiment of a method of obtaining human milkpermeate. As discussed above, donor milk is carefully analyzed for bothidentification purposes and to avoid contamination. The donor milk isfrozen and, when desired, thawed and pooled. It is then screened (step 1of FIG. 1), e.g., genetically screened, e.g., by polymerase chainreaction (PCR). Genetic screening is done to identify any contaminants,e.g., viral, e.g., HIV-1, HBV, and/or HCV. The milk then undergoesfiltering, e.g., through about a 200 micron filter (step 2), and heattreatment (step 3). For example, the composition can be treated at about63° C. or greater for about 30 minutes or more. In step 4, the milk istransferred to a separator, e.g., a centrifuge, to separate the creamfrom the skim. The skim can be transferred into a second processing tankwhere it remains at about 2 to 8° C. until a filtration step (step 5).Optionally, the cream separated from the skim in step 4, can undergoseparation again to yield more skim.

Following separation of cream and skim (step 4), a desired amount ofcream can be added to the skim, and the composition undergoes furtherfiltration (step 5), e.g., ultrafiltration, e.g., with a pore sizebetween 1-1000 Kda. This process concentrates the nutrients in the skimmilk by filtering out the what was previously thought to be generallywater or referred to as permeate. The present inventors have discovered,however, that the permeate retains a significant amount ofoligosaccharides and can itself be used, e.g., as a nutritionalsupplement or in other ways described herein.

Skim milk can undergo further processing for a human milk fortifier orstandardized human milk composition, as described, e.g., in U.S. Ser.No. 11/947,580. For example, the skim can be blended with cream toobtain bulk final product, pasteurized and processed for bioburden. Thepermeate is retained (step 6) and can be used as a nutritionalsupplement. FIG. 7 also depicts the production of a “permeate” and a“second permeate,” the latter produced by additional filtration of the“permeate.”

The permeate can be further processed, e.g., concentrated or dilutedand/or pasteurized. The permeate can be frozen and stored for futureuse. The permeate can be concentrated by reverse osmosis or dried usingtechniques familiar to those versed in the art. The resulting liquidproducts could then be pasteurized, sterile filtered, or subjected toother bioburden reduction steps. The product would then be filled intothe final product container.

The permeate can also be supplemented with vitamins and/or minerals,e.g., calcium, chloride, zinc, copper, iron, manganese, magnesium,phosphorus, potassium, sodium, selenium, chromium, molybdenum, iodine,taurine, carnitine, choline, vitamin A, vitamin B1, vitamin B2, vitaminB6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin,folic acid, pantothenic acid, niacin, and m-inositol. Vitamins andminerals are important, e.g., for proper nutrition and development of aninfant. Trace minerals are associated with cell division, immunefunction and growth. Some key trace minerals include copper, magnesium,and iron (which is important, e.g., for the synthesis of hemoglobin,myoglobin and iron-containing enzymes). Zinc is needed, e.g., forgrowth, for the activity of numerous enzymes, and for DNA, RNA andprotein synthesis. Copper is necessary for, e.g., the activity ofseveral enzymes. Manganese is needed, e.g., for the development of boneand cartilage and is important in the synthesis of polysaccharides andglycoproteins. Vitamin A is a fat-soluble vitamin essential for, e.g.,growth, cell differentiation, vision and proper functioning of theimmune system. Vitamin D is important, e.g., for absorption of calciumand to a lesser extent, phosphorus, and for the development of bone.Vitamin E (tocopherol) prevents peroxidation of polyunsaturated fattyacids in the cell, thus preventing tissue damage. Folic acid plays arole in, e.g., amino acid and nucleotide metabolism. Thus, the permeatecompositions can be supplemented with various minerals and/or vitamins,as desired.

Applications

The present disclosure features compositions that include human milkpermeate and methods of using such compositions. The permeatecompositions can be obtained, e.g., as described above by filteringliquid from skim milk. The compositions contain a significant number ofhuman oligosaccharides, fucosilated and/or sialilated as described inExample 1 below. Human milk also contains peptides and other smallmolecules that can also be present in the permeate, e.g., can beobtained by varying the pore size during ultrafiltration. Becausepermeate is derived from pooled human milk, it can contain more forms ofoligosaccharides than found in individual mother's milk. The permeatecompositions can be used alone or in conjunction with other milkcompositions, e.g., human milk compositions described in U.S. Ser. No.11/947,580 and non-human milk formulas. Permeate or human and non-humanmilk formulas supplemented with permeate can be administered to pre-termor full tem infants to, e.g., promote development of proper gut flora,treat conditions characterized by immunodeficiency, and treat or preventinfections. Permeate can also be administered to children and adultsalone or in conjunction with probiotics to promote beneficial gut flora,treat conditions characterized by immunodeficiency, and treat or preventinfections. Permeate compositions can be administered, e.g., orally ortopically to treat skin conditions.

Human Milk Oligosaccharides

Many factors can affect the clinical outcome of a newborn, e.g.,prematurely born, infant. Preterm infants have vulnerable immunesystems, immature digestive systems, and increased total caloric andspecific nutrient needs (when generally compared with full terminfants). Thus, nutrition provided to such infants is an importantfactor in their growth and development. Human milk has long beenrecognized as the ideal food for preterm and full term infants becauseof its nutritional composition and immunologic benefits. Not everymother, however, can or will breastfeed her baby (or use a breast pumpand store her milk). For example, mothers who have certain diseases,e.g., active tuberculosis, or are being administered radioisotopes,antimetabolites, or chemotherapy may not breast feed their infants. Inaddition, mother's own milk may not contain sufficient nutritionalcontent to sustain a preterm infant. Use of donor milk can also beproblematic, as such milk may not contain adequate nutrition for apreterm infant.

The present disclosure features permeate compositions that includeoligosaccharides from pooled human milk, and methods of using suchcompositions to benefit, e.g., of premature human infants, full terminfants, children, and adults.

Human milk oligosaccharides are carbohydrates that contain lactose atthe reducing end and, typically, a fucose or a sialic acid at thenonreducing end. (Morrow et al., J. Nutrit. 135:1304-07, 2005). Theseterminal sugars are the residues that most strongly influence theselective growth of bacteria and the interaction of oligosaccharideswith other molecules or cells, including bacterial pathogens in the gutlumen. For example, sialic acids are structural and functionalcomponents of brain gangliosides and have been implicated inneurological development of infants. Oligosaccharides can be free orconjugated as glycoproteins, glycolipids etc. and are classified asglycans. They constitute the third most numerous solid component ofhuman milk, after lactose and lipid (Morrow et al.). The majority ofthese milk oligosaccharides, however, are not digestable by infants andcan be found in infant feces largely intact. The present disclosuredescribes various uses of these oligosaccharides present in human milkpermeate (which, in fact, contains a wide variety of oligosaccharides asit is derived from a pool of donated milk), which was previously thoughtto be a waste product of processing human milk. The permeate orcompositions obtained from the permeate can be used (alone or incombination with human and non-human milk formulations or in combinationwith drugs or prebiotics), e.g., to promote beneficial gut flora, fightinfections, ameliorate symptoms of conditions characterized byimmunodeficiency, and provide additional nutrition. These compositionscan be beneficial to recipients of various ages: from preterm infantsthrough children to adults.

Gut Flora

Human gut flora, i.e., bacteria found in the gut, serves variousfunctions, including digestion of certain polysaccharides anddevelopment of the gut's mucosal immune system. Gut bacteria canstimulate the lymphoid tissue associated with the gut mucosa to produceantibodies to pathogens, leaving the helpful species alone, a tolerancedeveloped in infancy.

The gastrointestinal tract of a normal fetus is sterile. During birthand soon thereafter, the infant's gut is colonized by various bacteria.The sources of the bacteria can be the mother's body and/or theenvironment. After birth, various bacteria can be transferred from themother to the infant through suckling and touching. Most infant GItracts are initially colonized by large numbers of E. coli andStreptococci. Eventually, breast-fed babies become dominated bybifidobacteria, while formula-fed infants have a variety of bacteria,including Enterobacteriaceae, Enterococci, bifidobacteria, Bacteroides,and Clostridia in the gut. After the introduction of solid food andweaning, the microflora of breast-fed infants becomes similar to that offormula-fed infants. By about two years, the fecal microflora ofchildren is similar to that of adults.

Given the importance of gut flora, it is crucial to develop a beneficialbacterial population at birth and maintain it throughout childhood andadult life. The compositions and methods featured herein can help in theestablishment and maintenance of such beneficial gut flora.

The compositions that include human milk oligosaccharides, e.g., humanmilk permeate or compositions derived from human milk permeate, can beadministered to preterm infants, full term infants, children, andadults. They can be administered alone or in combination with othercompositions that aid in establishing beneficial gut flora, e.g.,probiotic bacteria or prebiotic plant polysaccharides, to form asymbiotic composition. They can be administered as part of human milkformulations (e.g., compositions described in application U.S. Ser. No.11/947,580) or non-human milk baby formulas. Without being bound bytheory, it is believed that the present permeate compositions canpromote colonization of the gut by lactobacilli and/or bifidobacteria.Both lactobacilli and bifidobacteria are known as probiotics—bacteriathat protect their host and prevent disease. Bifidobacteria areanaerobic bacteria that aid in digestion and have been associated withreduced incidence of allergies and cancer prevention. Lactobacilli arebacteria that convert lactose and other sugars to lactic acid and maypossess anti-inflammatory and anti-cancer properties.

Additionally, the human milk permeate or compositions derived from humanmilk permeate of the current invention containing human milkoligosaccharides can be administered to children or adults for thetreatment of inflammatory bowel disease. Inflammatory bowel disease is acollective term encompassing related, but distinct, chronic inflammatorydisorders of the gastrointestinal tract, such as Crohn's disease,irritable bowel disease, ulcerative colitis, indeterminate colitis,microscopic colitis, and collagenous colitis. It has been found thatpatients suffering from active Crohn's disease have significantly lessrecoverable bifidobacteria in their feces compared with healthyindividuals. This reduction in bifidobacteria numbers was observed to bedirectly correlated with decreased levels of β-D galactosidaseproduction and activity (Favier, C. et at, Dig. Dis. Sci. 1997;42:817-822). β-D galactosidase is an enzyme produced by bifidobacteria.These results support suggestions proposed in other studies that strainsof bifidobacteria may play important roles in maintaining a balancedhealthy intestinal microflora. Therefore, according to the presentinvention, the colony promotion of colonization of bifidobacteria in thegut by the oligosaccharide rich permeate of the current invention can beuseful in ameliorating the symptoms of inflammatory bowel disease.Similarly, the compositions of the current invention can be useful intreating pseudomembrenous colitis (also known as C. difficile-relatedcolits), a common complication of prolonged broad-spectrum antibiotictreatment that is due to overgrowth of normal flora by a toxin-producingstrain of C. difficile. The permeate compositions of the currentinvention can be administered before during or after any other treatmentfor inflammatory bowel disease including probiotic formulations,anti-inflammatory medications or immunomodulators.

Infections

Human milk oligosaccharides are thought to serve various anti-infectivefunctions, e.g., inhibit pathogen binding. Thus, the permeatecompositions featured herein and containing a wide variety of human milkoligosaccharides (as they are derived from pooled human milk) can beused to protect infants, children, and adults from various types ofinfection. For example, preterm and full term infants administered thepresent compositions can be protected from infectious diarrhea,necrotizing enterocolitis (NEC), respiratory tract infections,bacteremia, meningitis and others. Children and adults can likewisebenefit from administration of the compositions. The compositions can beadministered after an onset of an infection, to ameliorate and/oreliminate its symptoms or prior to infection (for individuals thought tobe at risk). The compositions can be administered via various means,including orally or topically (e.g., to treat skin infections and otherskin conditions).

In general, the permeate compositions featured herein can beadministered to subjects, e.g., human subjects of various ages, for anycondition or disorder that would be ameliorated or eliminated byapplication of oligosaccharides, peptides, or other small moleculesderived from human milk.

Kits

The present disclosure also features kits that include the human milkpermeate compositions described herein and containers for mixing thecompositions with raw human milk or non-human milk. The containers caninclude bottles, e.g., graduated bottles to assist in proper dilution,syringes, cans, and other containers known in the art.

The embodiments of the disclosure may be carried out in other ways thanthose set forth herein without departing from the spirit and scope ofthe disclosure. The embodiments are, therefore, to be considered in allrespects as illustrative and not restrictive.

Example 1 Collection and Analysis of Human Milk Permeate

Breast milk was collected from screened donors throughout the U.S. andstored at −20° C. for up to 12 months. Pooled donor human milk wasprocessed and various samples analyzed for their oligosaccharidecomposition. Pools from 20-50 donors were used to produce human milkfortifier compositions described in application U.S. Ser. No.11/947,580.

Before processing, a sample of initial pooled donor milk was set asidefor analysis (sample 49 in Table III). The pooled milk was screened,filtered, heat-treated, separated into skim and cream, and the skim wasultrafiltered. A portion of the filtered-out composition, the permeate,was set aside for analysis (sample 53 in Table IV). The skim was blendedwith cream and pasteurized. A portion of the bulk final product was setaside for analysis (samples 42, 48, and 54 in Tables I, II, and V,respectively).

Each milk sample that was set aside for analysis (0.5 mL) was dilutedwith 0.5 mL of pure water and centrifuged at 4000 rpm at 4° C. for 30minutes to separate fat. The permeate was analyzed undiluted. Thefat-free fraction was treated with 4 volumes (2:1) of achloroform-methanol solution (v/v). The emulsion was centrifuged at 3500rpm for 30 min at 4° C., and the lower chloroform layer and denaturedprotein were discarded. The upper layer was collected, two volumes ofpure ethanol were added and protein fraction was left to precipitate at4° C. overnight.

After protein separation by centrifugation at 3500 rpm for 30 min at 4°C., the limpid upper solution was collected and freeze-dried. Theresulting powder (freeze-dried oligosaccharide rich fraction) was usedfor oligosaccharide analysis. Oligosaccharides were reduced to alditolform using 1.0 M sodium borohydride in deionized water and incubated at42° C. overnight. After the reaction, oligosaccharides were purifiedfrom contaminants by solid-phase extraction using a nonporousgraphitized carbon cartridge (GCC-SPE). Nonporous graphitized carboncartridges (150 mg of bed weight, 4 mL tube size) for desalting werepurchased from Alltech (Deerfield, Ill., USA). Evaporation of solventswas performed using a speedvac centrifuge.

Solid Phase Extraction.

Prior to use, each GCC-SPE cartridge was washed with 3 column volumes of80% acetonitrile in 0.05% trifluoroacetic acid (TFA) (v/v) followed by 3column volumes of deionized water. After loading of the oligosaccharidemixture onto a cartridge, salts and residual peptides were removed bywashing with 8 cartridge volumes of deionized water. Theoligosaccharides were then eluted from the column using 10% acetonitrilein water (v/v) and 20% acetonitrile in water (v/v). Each fraction (6 mL)was collected and evaporated in vacuo prior to MS analysis.

Mass Spectrometric Analysis.

Matrix-assisted laser desorption/ionization (MALDI) Fouriertransform-ion cyclotron resonance mass spectrometry (FT ICR MS) wasperformed on an HiResMALDI (IonSpec Corp., Irvine, Calif.) equipped withan external MALDI source, a 7.0 Tesla superconducting magnet and apulsed Nd:YAG laser (355 nm). 2,5-Dihydroxybenzoic acid (DHB) was usedas a matrix (5 mg/100 μL in a solution of 50% acetonitrile/50% water(v/v)). The solution of oligosaccharide (1 μL) was applied to the MALDIprobe followed by addition of 0.01 M NaCl (0.5 μL) and the matrixsolution (1 μL). The sample was dried under a stream of air andsubjected to mass spectrometry.

The compositions of the various samples discussed above are presented inTables I-IV, and a summary is shown in FIG. 2. The experimentalmass:charge ratios (m/z expe) were matched to calculated mass:chargeratios (m/z cal) for known oligosaccharides in human milk. Massspectrometric profiles of specific samples are shown in Tables (humanmilk sample in Table III, bulk product in Table IV, and permeate inTable V). Mass spectrometric analyses of the five samples revealed ahighly complex mixture of oligosaccharides, typical of human milk, andvarying in size, composition, and abundance.

The oligosaccharides identified were from two main classes: (1) neutraloligosaccharides containing galactose, N-acetylglucosamine, lactose, andfuctose (Hex, HexNAc and Fuc); and (2) anionic oligosaccharidescontaining the same oligosaccharide compositions with the addition ofN-acetylneuraminic acid (NeuAC). A previous investigation (Ninonuevo etal., J. Agric. Food Chem. 2008, 54: 7471-7480) identified diversitybetween individuals in the total numbers and relative abundances ofspecific oligosaccharides in unfiltered milk. Thus, the milk samplesanalyzed (natural or concentrated) confirmed that all samples showostensibly most of the oligosaccharides previously found to be common inhuman milk from different donors.

TABLE I SAMPLE 42 (BULK FINAL PRODUCT) OLIGOSACCHARIDES m/z cxpc HcxHcxNAc Fuc NcuAC m/z cal Error Mode Form abund. 1389.477 4 2 2 01389.501 −0.0246 Na Alditol 100 1243.419 4 1 2 0 1243.443 −0.0239 NaAlditol 79.32 1754.611 5 2 3 0 1754.633 −0.0228 Na Alditol 36.361608.563 5 1 3 0 1608.576 −0.013 Na Alditol 22.4 1900.682 5 3 3 01900.691 −0.0095 Na Alditol 18.22 1535.545 4 3 2 0 1535.559 −0.0144 NaAlditol 17.4 878.3013 3 1 1 0 878.3111 −0.0098 Na Alditol 15.74 1097.3684 0 2 0 1097.385 −0.0176 Na Alditol 14.48 732.245 3 0 1 0 732.2532−0.0082 Na Alditol 13.02 1024.361 3 2 1 0 1024.369 −0.0083 Na Alditol6.88 1462.514 5 0 3 0 1462.518 −0.0041 Na Alditol 4.29 1556.519 4 1 2 11556.521 −0.0021 1 Na Alditol 3.48 2119.781 6 2 4 0 2119.766 0.0156 NaAlditol 2.72 2046.765 5 4 3 0 2046.749 0.0154 Na Alditol 2.24 1557.524 43 2 0 1557.541 −0.0168 1 Na Alditol 2.21

TABLE II SAMPLE 48 (BULK FINAL PRODUCT) OLIGOSACCHARIDES m/z expe HexHexNAc Fuc NeuAC m/z cal Error Mode Form abund. 1389.474 4 2 2 01389.501 −0.0274 Na Alditol 100 1754.609 5 2 3 0 1754.633 −0.0243 NaAlditol 15.63 1462.519 3 5 1 0 1462.543 −0.0239 Na Alditol 1.27 1243.4224 1 2 0 1243.443 −0.0212 Na Alditol 73.84 1405.476 5 1 2 0 1405.496−0.0202 Na Alditol 1.45 1900.672 5 3 3 0 1900.691 −0.0198 Na Alditol10.32 1535.54 4 3 2 0 1535.559 −0.0196 Na Alditol 23.25 1097.373 4 0 2 01097.385 −0.0129 Na Alditol 11.85 1608.567 5 1 3 0 1608.576 −0.0089 NaAlditol 6.79 1024.364 3 2 1 0 1024.369 −0.0047 Na Alditol 14.85 878.30753 1 1 0 878.3111 −0.0036 Na Alditol 24.05 2046.746 5 4 3 0 2046.749−0.0031 Na Alditol 1.28 732.2536 3 0 1 0 732.2532 0.0004 Na Alditol14.72 1462.519 5 0 3 0 1462.518 0.0012 Na Alditol 1.27 1081.392 3 1 2 01081.391 0.0015 Na Alditol 1.99 513.1818 2 1 0 0 513.1789 0.0029 NaAlditol 9.19 935.3364 3 0 2 0 935.3326 0.0038 Na Alditol 1.26 659.2407 22 0 0 659.2368 0.0039 Na Alditol 1.08

TABLE III SAMPLE 49 (INITIAL POOL OF DONOR MILK) OLIGOSACCHARIDES m/zexpe Hex HexNAc Fuc NeuAC m/z cal Error Mode Form abund. 1388.972 4 3 30 1388.99 −0.0202 Na Alditol 100 1754.608 5 2 3 0 1754.633 −0.0258 NaAlditol 5.1 1243.423 4 1 2 0 1243.443 −0.0207 Na Alditol 4.1 1900.671 53 3 0 1900.691 −0.0202 Na Alditol 1.48 1535.542 4 3 2 0 1535.559 −0.0169Na Alditol 5.9 1608.562 5 1 3 0 1608.576 −0.0139 Na Alditol 3.6 1097.3774 0 2 0 1097.385 −0.0084 Na Alditol 10.5 1024.364 3 2 1 0 1024.369−0.0054 Na Alditol 14.7 1462.513 5 0 3 0 1462.518 −0.0042 Na Alditol 1.2878.3073 3 1 1 0 878.3111 −0.0038 Na Alditol 26 732.2535 3 0 1 0732.2532 0.0003 Na Alditol 14.2 513.1826 2 1 0 0 513.1789 0.0037 NaAlditol 8.9 659.241 2 2 0 0 659.2368 0.0042 Na Alditol 1.66

TABLE IV SAMPLE 53 (PERMEATE) OLIGOSACCHARIDES m/z expe Hex HexNAc FucNeuAC m/z cal Error Mode Form abund. 1973.685 6 1 4 0 1973.708 −0.0229Na Alditol 100 2119.75 6 2 4 0 2119.766 −0.0152 Na Alditol 89.8 1608.5645 1 3 0 1608.576 −0.0116 Na Alditol 69.33 1754.626 5 2 3 0 1754.633−0.0077 Na Alditol 54.88 1827.651 6 0 4 0 1827.65 0.0009 Na Alditol41.26 2265.835 6 3 4 0 2265.824 0.0119 Na Alditol 34.5 1462.52 5 0 3 01462.518 0.0022 Na Alditol 28.01 1900.709 5 3 3 0 1900.691 0.0177 NaAlditol 15.16 2484.931 7 2 5 0 2484.898 0.0334 Na Alditol 15.12 2338.8687 1 5 0 2338.84 0.028 Na Alditol 14.2 1243.447 4 1 2 0 1243.443 0.0039Na Alditol 13.39 1389.508 4 2 2 0 1389.501 0.0069 Na Alditol 11.41732.2539 3 0 1 0 732.2532 0.0007 Na Alditol 8.37 2631.035 7 3 5 02630.956 0.0792 Na Alditol 4.85 2192.827 7 0 5 0 2192.782 0.0447 NaAlditol 4.82 2411.932 6 4 4 0 2411.881 0.0504 Na Alditol 3.39 2704.042 81 6 0 2703.972 0.0697 Na Alditol 2.85 1556.543 4 1 2 1 1556.521 0.0227Na Alditol 2.67 2850.121 8 2 6 0 2850.03 0.0908 Na Alditol 2.16 570.20122 0 1 0 570.2004 0.0008 Na Alditol 1.94 1097.392 4 0 2 0 1097.385 0.0063Na Alditol 1.82 2557.988 8 0 6 0 2557.914 0.0739 Na Alditol 1.281300.476 4 0 3 0 1300.465 0.011 Na Alditol 1.19 659.2383 2 2 0 0659.2368 0.0015 Na Alditol 1.16 513.1802 2 1 0 0 513.1789 0.0013 NaAlditol 1.01

TABLE V SAMPLE 54 (BULK FINAL PRODUCT) OLIGOSACCHARIDES m/z expe HexHexNAc Fuc NeuAC m/z cal Error Mode Form abund. 513.1576 2 1 0 0513.1789 −0.0213 Na Alditol 12.82 659.2137 2 2 0 0 659.2368 −0.0231 NaAlditol 1.38 570.1783 2 0 1 0 570.2004 −0.0221 Na Alditol 4.24 878.27823 1 1 0 878.3111 −0.0329 Na Alditol 20.97 1024.338 3 2 1 0 1024.369−0.0309 Na Alditol 8.82 732.2245 3 0 1 0 732.2532 −0.0287 Na Alditol 16935.3093 3 0 2 0 935.3326 −0.0233 Na Alditol 1.16 1243.39 4 1 2 01243.443 −0.0536 Na Alditol 76.72 1389.445 4 2 2 0 1389.501 −0.0567 NaAlditol 100 1535.524 4 3 2 0 1535.559 −0.0348 Na Alditol 19.2 1097.334 40 2 0 1097.385 −0.0511 Na Alditol 14.86 1608.547 5 1 3 0 1608.576 −0.029Na Alditol 19.86 1754.592 5 2 3 0 1754.633 −0.0416 Na Alditol 36.71900.669 5 3 3 0 1900.691 −0.0225 Na Alditol 24.32 2046.773 5 4 3 02046.749 0.0236 Na Alditol 4.32 1462.497 5 0 3 0 1462.518 −0.0204 NaAlditol 3.8 1973.729 6 1 4 0 1973.708 0.0213 Na Alditol 1.66 2119.792 62 4 0 2119.766 0.0261 Na Alditol 4.15 2265.855 6 3 4 0 2265.824 0.0314Na Alditol 4.45 2411.941 6 4 4 0 2411.881 0.0596 Na Alditol 1.3 m/zexpe: Mass/charge experimental, m/z cal: mass/charge calculated, Hex:hexose; HexNAc: N-acetylhexosamine; Fuc: fucose; NeuAc:N-acetyl-neuraminic acid or sialic acid;

It appears that processed human milk product (fortifier or samples 42,48, and 54) and permeate (sample 53) contain the full range ofoligosaccharides commonly found in human milk. The selection and amountsof the individual oligosaccharides present in these samples reflect therelative amounts in the donor pool which average out the variationacross individual donors.

Thus, according to the present invention, the permeate is a valuablesource of human milk oligosaccharides that can be used as a nutritionalor immunologic supplement for preterm infants, infants, children, andadults.

Example 2 Oligosaccharide Profile of Pooled Permeate Samples

Six pooled permeate samples were analyzed for specific oligosaccharidecontent (Table VI). Each of the pooled permeate samples were derivedfrom pooled milk received from multiple donors by the method discussedabove in Example 1. The collected samples of permeate were analyzed bymass spectrometry by the method described above in Example 1, to detectthe presence (1) or absence (0) of the specific oligosaccharidesindicated. The oligosaccharides are represented in Table VI by theirmass/charge ratios.

TABLE VI Per- Per- Per- Olgios meate meate meate Permeate Permeate m/zPermeate Sample Sample Sample Sample Sample expe Sample #5 #11 #23 #28#47 #53 513 0 0 1 0 1 0 732 1 1 1 1 1 1 878 1 1 1 1 1 1 935 0 0 1 0 0 11024 1 1 1 1 1 0 1097 1 1 1 1 1 1 1243 1 1 1 1 1 1 1389 1 1 1 1 1 1 14621 1 1 0 1 1 1535 0 0 1 0 1 0 1556 0 0 0 0 0 1 1608 1 0 1 1 1 1 1754 0 11 1 0 1 1827 1 0 0 0 0 1 1900 0 1 0 0 0 1 1973 1 0 0 1 0 1 2119 1 1 0 10 1 2192 0 0 0 0 0 1 2264 0 0 0 0 0 1 2338 0 0 0 0 0 1 2411 0 0 0 0 0 12485 0 0 0 0 0 1 2631 0 0 0 0 1 1 = oligosaccharide present in thesample 0 = oligosaccharide not detectable in the sample

It appears as though, while there is overlap between the samples withrespect to certain oligosaccharides (i.e. oligosaccharides 732, 878,1097, 1243 and 1389 are present in all six samples), there are also manyoligosaccharides present in certain samples while not present in othersamples (i.e. oligosaccharide 935 is present in permeate numbers 23, and53 but not permeate numbers 5, 11, 28 or 47). Therefore, additionalpooling of the pooled permeate samples may be necessary in order toobtain a composition with substantially all or all oligosaccharidespecies represented.

Example 3 Administration of Permeate Compositions

As discussed above, it is believed by the inventors that human milkpermeate compositions from pooled milk can provide a wide variety ofnutritional and immunologic benefits, as they are a source of a widervariety of oligosaccharides than those found in individual mother'smilk. They can also contain various types of peptides and otherbeneficial small molecules.

The isolated human milk permeate compositions will be administered toinfants, both preterm and full term infants, to promote theirdevelopment of proper gut flora, ameliorate and/or eliminate infections.The study will investigate infants divided into 5 arms including infantsreceiving:

1. Mother's milk plus fortifier

2. Cow's milk plus permeate

3. Cow's milk plus strain 1 of bifidobacter

4. Cow's milk plus strain 2 of bifidobacter

5. Cow's milk plus non-milk derived fructose oligosaccharide

Composition of infant feces will be monitored to determine the contentof their gut flora. Infants suffering from infections caused bypathogens, e.g., bacteria, bacterial toxins, and viruses willparticularly benefit from oral administration of the permeatecompositions. It is expected that the gut flora from infants receivingcow's milk plus permeate will most closely mimic the gut flora frominfants receiving mother's milk plus fortifier.

Other variations and embodiments of the invention described herein willnow be apparent to those of ordinary skill in the art without departingfrom the scope of the invention or the spirit of the claims below.

1-11. (canceled)
 12. A method of making a concentrated form of humanmilk permeate comprising human milk oligosaccharides, the methodcomprising: (a) obtaining human milk; (b) separating the milk into creamand skim; (c) filtering the skim to obtain a permeate; (d) retaining thepermeate; and (e) concentrating the permeate. 13-15. (canceled)
 16. Themethod of claim 12 wherein the concentrating step in step (e) comprisesreverse osmosis.
 17. The method of claim 12, further comprising addingvitamins and minerals to the permeate after step (d)
 18. The method ofclaim 12 wherein the filtering comprises ultrafiltering.
 19. The methodof claim 12 further comprising (f) reducing the bioburden.
 20. Themethod of claim 19 wherein reducing the bioburden comprisespasteurization or sterile filtration. 21-63. (canceled)
 64. The methodof claim 12 wherein the human milk is pooled from two or more donorsprior to step (b).
 65. The method of claim 64 wherein the human milk ispooled to arrive at 75-2000 liters/lot of human milk.
 66. The method ofclaim 18 wherein the ultrafiltering comprises filtering through filtersabout 1 Kda-1,000 Kda in size.
 67. The method of claim 64 wherein thehuman milk is frozen prior to pooling.
 68. The method of claim 64wherein the pooled human milk is genetically screened to identifycontaminants.
 69. The method of claim 68 wherein the contaminants areviral contaminants.
 70. The method of claim 69, wherein the viralcontaminants are HIV-1, HBV, and/or HCV.
 71. The method of claim 68wherein the pooled human milk is genetically screened by polymerasechain reaction (PCR).
 72. The method of claim 64 wherein the pooledhuman milk is filtered through a 200 micron filter prior to step (b).73. The method of claim 64 wherein the pooled human milk is heated toabout 63° C. or greater for at least about 30 minutes prior to step (b).